The present invention concerns a method for expanding stem cells from blood, particularly but not only peripheral blood, of adult mammals, and the relative application in the medical field, in particular in the veterinary field, for the treatment of lesions, chronic and/or acute inflammatory pathologies, and neurological and neurodegenerative pathologies. Here, and hereafter in the description, and as is known in literature, the terms “expanding” and “expansion” refer to the process of increasing the number of cells either by cell division or, as in the specific case described and claimed, by “de-differentiation”, that is, the process by which the cells present in the blood are transformed into stem cells following suitable in-vitro treatment, as will be seen hereafter.
In recent years the use of stem cells in therapy has had great approval due to the successes obtained in treating various pathologies which were previously thought incurable. However, processes known until now for obtaining stem cells have been laborious and expensive.
Pluripotent stem cells (PSC) are a source available not only for research but also for the creation of drugs and for transplants (A. J. Wagers et al., “Cell fate determination from stem cells”; Gene Therapy; 9; 606-612 (2002); L. G. Griffith et al., “Tissue Engineering—Current Challenges and Expanding Opportunities”; Science; 295; 1009 (2002)).
There are two ample categories of stem cells: embryonic and adult. The former are derived from embryos, and more exactly from 8-day blastocysts, whereas adult stem cells may be obtained mainly from bone marrow, adipose or muscular tissue, or from peripheral blood.
The definition of stem cell is constantly evolving and, at the moment, there is no general consensus or standard method to isolate or identify them. For all these cells, embryonic (ES) and adult, both hematopoietic (HSC) and mesenchymal (MSC) (M. Kuwana et al., “Human circulating CD14+ monocytes as a source of progenitors that exhibit mesenchymal cell differentiation”; J. Leuk Biol; 74; 833-845 (2003)), different genetic markers have been identified, of which some are common to many cell types. (See, for example M. Condomines et al., “Functional Regulatory T Cells Are Collected in Stem Cell Autographs by Mobilization with High-Dose Cyclophosphamide and Granulocyte Colony-Stimulating Factor”; J. Immunology; 176: 6631-6639 (2006); W. J. Kang et al., “Tissue Distribution of 18F-FDG-Labeled Peripheral Hematopoietic Stem Cells After Intracoronary Administration in Patients with Myocardial Infarction”; J. Nucl Med.; 47:1295-1301 (2006); Y. Zhao et al., “A human peripheral blood monocyte-derived subset acts as pluripotent stem cells”; PNAS; vol. 100, No. 5; 2426-2431 (2003); and M. Rabinovitch et al., “Cell Shape Changes Induced by Cationic Anesthetics”; J. Experimental Medicine; 143; 290-304 (1976)).
In particular, Zhao Y. et al. (“A human peripheral blood monocyte-derived subset acts as pluripotent stem cells”; PNAS; vol. 100, No. 5; 2426-2431 (2003) and WO 2004/043990) discloses a method for preparing monocyte-derived stem cells which includes the steps of isolating a peripheral-blood monocyte, contacting it with a mitogenic component, and subsequently culturing the peripheral-blood monocyte under conditions suitable for the propagation of the cells.
This method, which requires a first step of isolating the monocyte and a subsequent expansion step in a culturing media, requires very long times, on the order of 15-20 days, to obtain a significant number of stem cells. By this method, it is not possible to obtain stem cells of the totipotent type, that is, cells that are non-specialized and suitable to be directly inoculated into the patient after a very short time from the first drawing.
Numerous scientific works describe stem cells' ability to regenerate different types of lesions by regenerating tissues that are mechanically damaged or are damaged by various pathologies, thus eliminating at the root the causes that generated the pathology and not simply acting on the effects thereof.
At the moment, research is more oriented toward the use of stem cells isolated from embryonic tissue, fetuses, or the umbilical cord, but this work is raising various legal and ethical questions. Above all, as of today the use of these cells brings various contraindications, such as risk of infection, risk of rejection if transplanted and, in horses, the risk of onset of teratomas.
To obviate these problems, it has therefore been contemplated to use in the “in vivo” therapy autologous stem cells isolated preferably from bone marrow, adipose tissue or peripheral blood. These methods, starting from adult stem cells, provide a step of differentiation “in-vitro” (or “ex vivo”) of the stem cells in the cell line desired by means of specific differentiation induction factors, and a subsequent step of “in vivo” transplantation of the differentiated cell line obtained. In these methods, the limit is due to the fact that observable rejection phenomena occur because the differentiated cells re-introduced into the patient are not recognized as self-cells, but lose the self-recognition factors during the differentiation step induced in-vitro.
In humans, taking stem cells from peripheral blood entails purifying them through a process called “aphaeresis” or “leucophaeresis”. In practice, the cells are extracted from the blood, collected, and then inoculated into patients immediately after chemo- or radio therapy. In aphaeresis, which lasts from 6 to 8 hours, the blood is taken from the vein of an arm or a vein in the neck or the chest, and made to pass through a machine which removes the stem cells. The blood, thus purified, returns to the patient, while the cells collected are preserved by means of refrigeration in liquid nitrogen (M. Condomines et al., “Functional Regulatory T Cells Are Collected in Stem Cell Autographs by Mobilization with High-Dose Cyclophosphamide and Granulocyte Colony-Stimulating Factor”; J. Immunology; 176: 6631-6639 (2006); W. J. Kang et al., “Tissue Distribution of 18F-FDG-Labeled Peripheral Hematopoietic Stem Cells After Intracoronary Administration in Patients with Myocardial Infarction”; J. Nucl Med.; 47:1295-1301 (2006)). This technique is not only painful, but also extremely stressful for the patient. Furthermore, it is impracticable for animals of either small or large size; above all, the technique does not provide a real discrimination and/or purification of the stem cells circulating.
At present, in veterinary science, stem cells are used successfully mainly in the reconstruction of tendons and ligaments with lesions. The main techniques for purification include:
use of growth factors or platelet derivatives (TGF-B, VEGF), but the economic costs of extracting these are prohibitive (M. Hou et al., “Transplantation of mesenchymal stem cells from human bone marrow improves damaged heart function in rats”; International Journal of Cardiology; 115; 220-228 (2006));
isolation of stem cells taken from bone marrow. This technique provides for purification and then use for therapy of only 15% of the cells contained in the material extracted;
isolation of stem cells taken from adipose tissue. This technique, which requires the prior surgical removal of considerable quantities of tissue from the donor animal, does not allow for intravenous administration;
IGF-1 (insulin-like growth factor 1), known as Tendotrophin (J. Fiedler et al., “IGF-I and IGF-II stimulate directed cell migration of bone-marrow-derived human mesenchymal progenitor cells”; Biochemical and Biophysical Research Communications; 345; 1177-1183 (2006));
UBM (urinary bladder matrix), a derivative from the pig containing cytokines (but not nucleate cells), which induces the cicatrization of the wound but not the regeneration of the zone with lesions (Y. S. Zhang et al., “Preliminary research on preparation of porcine bladder acellular matrix graft for tissue engineering applications”; Zhonghua Yi Xue Za Zhi; 85(38); 2724-2727 (2005)).
In the light of all the above, it is obvious that methods are needed for the expansion and purification of adult stem cells from easily accessible sources which must also provide for obtaining stem cells suitable for use as medication in the medical-veterinary field. Once administered in the mammal, such cells do not give rise to phenomena of rejection and are easy to preserve.
There is also an obvious need for obtaining stem cells of the pluripotent and totipotent type, that is, non-specialized cells, which can be inoculated directly into the patient with much shorter production times than those provided at present.